Human exhaled aerosol droplet biomarker system and method

ABSTRACT

A system and method for detecting a biomarker in exhaled breath condensate nanodroplets comprises noninvasively collecting exhaled breath condensate nanodroplets of a subject, and analyzing said nanodroplets utilizing immuno-quantitative polymerase chain reaction to detect one or more target biomarkers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on U.S. Provisional Patent Application Ser.No. 61/696,641, filed Sep. 4, 2012, and Application Ser. No. 61/726,423,filed Nov. 14, 2012, which applications are both incorporated herein byreference in their entireties and to which priority is claimed.

FIELD OF THE INVENTION

The present invention relates to a system and method for detection ofnonvolatile biomarkers in exhaled breath nanodroplets.

BACKGROUND OF THE INVENTION

Chronic lung diseases are a significant health and economic burdenworldwide. For example, chronic obstructive lung disease (COPD) is thefourth leading cause of death in adults (Rennard S I (1998) “COPD:overview of definitions, epidemiology, and factors influencing itsdevelopment,” Chest. 113:235 S-241S), and lung cancer has the highestmortality of all cancers in both men and women (Alberg A J and Samet J M(2003) “Epidemiology of lung cancer,” Chest. 123:21 S-49S). The commonrisk factor for both is cigarette smoking. However, only 10-15% ofsmokers develop COPD and/or lung cancer. Non-invasive efforts toidentify biomarkers for such conditions have not been efficient orclinically effective.

Conventional methodologies typically provide for measuring biomarkers inlungs via invasive procedures such as bronchoscopy to obtain samples,carrying the associated costs, discomfort, and risks. Such biomarkersare needed for identifying the environmental factors in the generationand natural history of chronic lung diseases, and would allow forreliably following molecular events that are currently beyond detectionusing conventional methodologies.

Exhaled biomarkers could serve as a molecular and genetic signature,opening the doors for personalized medicine. Exhaled breath is anaerosol consisting mostly of water vapor, with smaller amounts ofvolatile, semi-volatile, and non-volatile molecules derived from theupper and lower portions of the respiratory system (Effros R M et al.(2005) “Epithelial lining fluid solute concentrations in chronicobstructive lung disease patients and normal subjects,” J. Appl.Physiol. 99:1286-1292; Horvath I et al. (2005) “Exhaled breathcondensate: methodological recommendations and unresolved questions,”Eur. Respir. J. 26:523-548).

Cytokines are small, water-soluble signaling proteins produced by cellsof the immune system to modulate responses of the immune system such asinflammation. Since inflammation is an underlying condition of manychronic diseases, exhaled cytokines may be considered biomarkers ofpulmonary inflammation that could indicate the presence of lung diseasesor provide information regarding the current status of the lungs. Assuch, non-invasive monitoring of lung inflammation through detection andmeasurement of cytokines in exhaled breath samples would be a promisingnew approach aimed at addressing the need for an improved understanding,treatment and management of chronic respiratory diseases such as lungcancer, asthma and COPD.

Thus, there has been great interest in the study of exhaled breathcondensate (EBC), and in techniques for the collection and analysis ofnon-volatile compounds (e.g. cytokines) present in the respiratorylining fluid (RLF). Studies of exhaled breath suggest that humansgenerate fine particles during tidal breathing, but little is known oftheir origin in the respiratory system. Older studies of exhaled breathprimarily detected particles larger than 1 μm due to less sensitivetechniques, such as counting particles in photographs of coughs andsneezes (Jennison M W “Atomizing of Mouth And Nose Secretions into theAir as Revealed by High-Speed Photography, in Aerobiology Publication,”Washington, D.C.: American Association for the Advancement of Science,p. 106), culturing of indicator bacteria exhaled and impacted on plates(Duguid J (1945) “The numbers and the sites of origin of the dropletsexpelled during expiratory activities,” Edinburgh Med. J. 52:385-401),and counting slides or filters of exhaled dye droplets under amicroscope (Id.; Loudon R G and Roberts R M (1967) “Droplet expulsionfrom the respiratory tract,” Am Rev Respir. Dis. 95:435-442). In sucholder studies, particles were typically detected only during coughs andsneezes, and not in breath exhaled during tidal breathing.

In more recent studies, it has been shown that approximately 98% ofparticles produced during tidal breathing are under 1 μm (Fairchild C Iand Stampfer J F (1987) “Particle concentration in exhaled breath,” Am.Ind. Hyg. Assoc. J. 48:948-949; Papineni R S and Rosenthal F S (1997)“The size distribution of droplets in the exhaled breath of healthyhuman subjects,” J. Aerosol Med. 10:105-116; Edwards D A et al. (2004)“Inhaling to mitigate exhaled bioaerosols,” Proc. Natl. Acad. Sci. USA101:17383-17388; Morawska L et al. (2008) “Size distribution and sitesof origin of droplets expelled from the human respiratory tract duringexpiratory activities,” J. Aerosol. Sci. 40:256-269). For example, in aprevious study of subjects infected with influenza, it was found thatthe subjects produced 67 to 8500 particles per liter of air, and that87% of the particles were under 1 μm (Fabian P et al. (2008) “Influenzavirus in human exhaled breath: an observational study,” PLoS ONE3:e2691).

Such droplets can be generated by shear forces produced by air flowacting on the airway lining fluid and entraining particles composed ofmucus, surfactant, and pathogens (King M et al. (1985) “Clearance ofmucus by simulated cough,” J. Appl. Physiol. 58:1776-1782; Moriarty J Aand Grotberg J B (1999) “Flow-induced instabilities of a mucus-serousbilayer,” J. Fluid Mech. 397:1-22), especially during cough (Leith D etal. (1986) “Cough” in MJ Macklem (ed). Handbook of Physiology, TheRespiratory System, Section 3, Vol. III, Part 1, Bethesda, Md.: AmericanPhysiological Society, pp. 315-336). It has been hypothesized thatdroplets are produced from the destabilization of the lining fluidduring the reopening of collapsed small airways and alveoli duringbreathing (Edwards D A et al. (2004), supra., Proc. Natl. Acad. Sci. USA101:17383-17388). Another study found that exhaled particleconcentrations increased 4- to 18-fold when inhaling deeply and rapidlyafter a deep exhalation, hypothesizing that the opening of airways andalveoli blocked by fluid during inhalation is a significant source ofparticles (Johnson G R and Morawska L (2009) “The mechanism of breathaerosol formation,” J. Aerosol Med. Pulm. Drug Deliv. 22:229-237).Identifying the origin of these particles is important when interpretingstudies of exhaled breath biomarkers, including cytokines (Shahid S K etal. (2002) “Increased interleukin-4 and decreased interferon-gamma inexhaled breath condensate of children with asthma,” Am. J. Respir. Crit.Care Med., 165:1290-1293; Garey K W et al. (2004) “Markers ofinflammation in exhaled breath condensate of young healthy smokers,”Chest. 125: 22-26; Rosias P P et al. (2004) “Childhood asthma: exhaledmarkers of airway inflammation, asthma control score, and lung functiontests,” Pediatr. Pulmonol. 38:107-114; Carpagnano G E et al. (2002)“Interleukin-6 is increased in breath condensate of patients withnon-small cell lung cancer,” Int. J. Biol. Markers, 17:141-145; Leung TF et al. (2004) “Increased macrophage-derived chemokine in exhaledbreath condensate and plasma from children with asthma,” Clin ExpAllergy, 34:786-791; and Rosias P et al. (2004) “Exhaled breathcondensate: a space odessey, where no one has gone before,” Eur. Respir.J. 24:189-190), metals (Broding H C et al. (2009) “Comparison betweenexhaled breath condensate analysis as a marker for cobalt and tungstenexposure and biomonitoring in workers of a hard metal alloy processingplant,” Int. Arch. Occup. Environ. Health. 82:565-573; Goldoni M et al.(2008) “Chromium in exhaled breath condensate and pulmonary tissue ofnon-small cell lung cancer patients,” Int. Arch. Occup. Environ. Health,81:487-493; Mutti A et al. (2006) “Exhaled metallic elements and serumpneumoproteins in asymptomatic smokers and patients with COPD orasthma,” Chest. 129:1288-1297), and pathogens such as viruses (Fabian Pet al. (2008), supra., PLoS ONE 3:e2691; Huynh K N et al. (2008) “A newmethod for sampling and detection of exhaled respiratory virusaerosols,” Clin. Infect. Dis. 46:93-95) and bacteria (Fennelly K P etal. (2004) “Cough-generated aerosols of Mycobacterium tuberculosis: anew method to study infectiousness,” Am. J. Respir. Crit. Care Med.169:604-609).

Collection of EBC samples non-invasively may be accomplished throughmeans whereby a subject breathes normally into a chilled collectiondevice that condenses and collects fluid samples. EBC samples consist ofa mixture of three main components (Horvath I et al. (2005), supra.,Eur. Respir. J. 26:523-548). The most abundant component (99%) of EBCsamples is liquid water formed from the condensation of water vaporpresent in the warm exhaled air, saturated with water vapor as it leavesthe respiratory tract. The second and third components of EBC samplesare water-soluble volatile and non-volatile particles that areaerosolized from the respiratory lining fluid and are present insignificantly smaller amounts than the water component of EBC samples(Horvath I et al. (2005), supra., Eur. Respir. J. 26:523-548; KietzmannD et al. (1993) “Hydrogen peroxide in expired breath condensate ofpatients with acute respiratory failure and with ARDS,” Intensive CareMed. 19:78-81; Effros R M et al. (2002) “Dilution of respiratory solutesin exhaled condensates,” Am. J. Respir. Crit. Care Med. 165:663-669;Horvath I et al. (2009) “Exhaled biomarkers in lung cancer,” Eur.Respir. J. 34:261-275; Kazani S and Israel E (2010) “Exhaled breathcondensates in asthma: diagnostic and therapeutic implications,” J.Breath Res. 4:047001; Loukides S et al. (2011) “Exhaled breathcondensate in asthma: from bench to bedside,” Curr. Med. Chem.18:1432-1443).

Unfortunately, the significant amount of liquid water present in EBCsamples dilutes the inherently low concentrations of non-volatilebiomarkers to levels that are at or below the detection threshold ofmethodologies utilizing conventional assays. Moreover, the inefficientcollection of exhaled, nonvolatile submicron particles usingconventional EBC collection methods, combined with assay sensitivitylimitations currently being used, creates significant problems withreproducibility and validity of biomarker measurements (Horvath I et al.(2005), supra., Eur. Respir. J. 26:523-548; Kazani S and Israel E (2010)“Exhaled breath condensates in asthma: diagnostic and therapeuticimplications,” J. Breath Res. 4:047001; Loukides S et al. (2011),supra., Curr. Med. Chem. 18:1432-1443; Sack U et al. (2006) “Multiplexanalysis of cytokines in exhaled breath condensate,” Cytometry A.69:169-172; Bayley D L et al. (2008) “Validation of assays forinflammatory mediators in exhaled breath condensate,” Eur. Respir. J.31:943-948; Sapey E et al. (2008) “The validation of assays used tomeasure biomarkers in exhaled breath condensate,” Eur. Respir. J.32:1408-1409). For example, the aerosol particle collection efficiencyof conventional EBC devices is typically less than 25%.

As a result, conventional systems and methods for biomarker collectionand analysis fail to provide sufficient sensitivity and efficiency fordetecting cytokines and other non-volatile biomarkers in EBC. Theavailability of an effective, non-invasive system and methodology ableto detect trace amounts of target biomarker(s) would open a new world ofpossibilities to the diagnosis and management of lung diseases anddisorders.

SUMMARY OF THE INVENTION

The present invention is directed to an integrated system and method fordetection of nonvolatile biomarkers in exhaled breath nanodroplets ofrespiratory lining fluid from the distal lung. The disclosed system farexceeds the sensitivity and reliability of conventional exhaled breathcondensate collection and assay systems, and includes various innovativecomponents and techniques. The disclosed systems and methodologies aresuitable for the diagnosis and management of various diseases anddisorders, such as lung cancer, asthma, COPD, tuberculosis, influenza,and HIV/AIDS related respiratory infections.

According to one aspect, specialized breathing maneuvers are utilized(exhalation to residual volume while supine), which increase output ofrespiratory droplets of distal airway lining fluid containingnonvolatile biomarkers, rather than normal tidal breathing used inconventional methods.

According to another aspect, collection using air pollution techniquesis utilized for collection of particle matter (PM) with a range of sizesfrom nanodroplets through PM 10, rather than condensation of water vaporfrom exhaled breath as in conventional methods.

According to another aspect, efficient recovery of labile proteins fromthe collection device is provided, by direct impaction in a liquid andextraction filters. In one implementation, the disclosed system providesfor high efficiency nanodroplet collection by impaction into a liquidmedia. A collection device is provided which maximizes impaction viaprecise air flow control, combined with collection of the droplets onfilters.

According to another aspect, immuno-quantitative polymerase chainreaction (IqPCR) assay is utilized to detect as few as 100 molecules ofbiomarker.

According to another aspect, optical particle counts are utilized todetermine the volume of respiratory fluid droplets generated by eachsubject, allowing direct standardization of the biomarker results interms of concentration in lung lining fluid.

The present invention provides for a method for detecting a biomarker inexhaled breath condensate nanodroplets, comprising the steps of:noninvasively collecting exhaled breath condensate (EBC) nanodropletsfrom a subject; and analyzing said nanodroplets utilizingimmuno-quantitative polymerase chain reaction (IqPCR) to detect one ormore target biomarkers.

In one implementation, the biomarkers are associated with a respiratorydisease, disorder or infection, such as lung cancer, asthma, chronicobstructive pulmonary disease, tuberculosis, influenza, a humanimmunodeficiency virus (HIV) related infection, and an acquired immunedeficiency syndrome (AIDS) related infection. The biomarkers may becytokines (e.g., such as IFN-gamma, IL-1 beta, IL-7, IL-8, IL-13, andTNF-alpha).

In one implementation, the nanodroplets are impacted into a liquidmedium by a collection device during the collection process. The volumeof nanodroplets generated by the subject is determined, and aconcentration of the detected biomarkers within the collectednanodroplets may then be standardized. In some implementations, thesubject is disposed in a supine position during the collection processin order to increase nanodroplet production.

The present invention also provides for a system for detectingnonvolatile biomarkers in exhaled breath nanodroplets. The systemcomprises a collection device configured to noninvasively collectexhaled breath condensate nanodroplets from a subject, andimmuno-quantitative polymerase chain reaction (IqPCR) assay fordetecting one or more target biomarkers in the nanodroplets.

In one implementation, the IqPCR assay comprises a microbead having anantibody immobilized thereon, which binds to a particular targetbiomarker. The IqPCR exhibits at least a 10-fold increase, morepreferably at least a 1000-fold increase, in sensitivity in detection ofsaid target biomarkers as compared to enzyme-linked immuno-sorbent assay(ELISA).

The present invention also provides for a microfluidic device fordetecting one or more biomarkers in nanodroplets. The microfluidicdevice comprises a substrate comprising a microchannel having an inletand an outlet, and a microbead-antibody complex disposed within saidmicrochannel, said microbead-antibody complex configured to detect saidbiomarker.

In one implementation, the microfluidic device comprises a thermocyclerdisposed on the substrate and in fluid communication with themicrochannel. The thermocycler comprises an IqPCR assay utilizing aDNA-labeled recognition antibody adapted to target biomarkers bound tothe microbead-antibody complex. In some embodiments, the microfluidicdevice is a multiplexed IqPCR device configured to simultaneously detectmultiple biomarkers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a device according to the present invention forcollecting particles from exhaled breath condensate.

FIG. 2 is a schematic of IqPCR utilizing microbeads for detecting traceamounts of a biomarker.

FIG. 3 is a schematic of a multiplexed immunoassay within a microfluidicchannel of a microfluidic device according to the present invention.

FIG. 4 is a schematic of a microfluidic droplet IqPCR for multiplexeddetection of trace biomarkers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a non-invasive diagnostic systemand method to quantitatively measure nonvolatile biomarkers (e.g.proteins such as cytokines, DNA, lipids, etc.) dissolved in respiratoryfluid of the lower respiratory tract. The present invention is alsodirected to a microfluidic multiplexed IqPCR device.

The detection of low concentrations of biomarkers with accuracy andsensitivity in test samples poses a significant problem and is notfeasible with conventional immunoassays such as enzyme-linkedimmuno-sorbent assay (ELISA). By combining immunoassay with nucleic acidamplification, the disclosed methodologies overcome the sensitivitylimitations of such conventional techniques. The amplification providedby IqPCR enables the detection of single nucleic acid molecules. In oneimplementation, a DNA probe hybridized to its template providesexceptional sensitivity and target specificity. IqPCR allows fordetection of biological molecules at concentrations as low as 1femtogram/mL, or even in the attogram level, thus providing a 10- to1000-fold increase in sensitivity over conventional methods such asELISA.

The detection of nucleic acids can be achieved at levels of a fewmolecules using IqPCR. In contrast, the analysis of proteins usingconventional methods, such as ELISA, hardly surpasses sensitivity levelsbelow 1×10⁻¹⁸ mol of the antigen. By combining the enormousamplification power of PCR with antibody-based immuno-assays,sensitivity is substantially enhanced (e.g., see Adler M et al. (2008)“Sensitivity by combination: immuno-PCR and related technologies,”Analyst, 133:702-718). IqPCR is based on chimeric conjugates of specificantibodies and nucleic acid molecules. The nucleic acid molecules areused as markers to be amplified by PCR for signal generation. In oneimplementation, methodologies for conjugating antibodies with DNA suchas disclosed by Adler M et al. (2008), supra., Analyst, 133:702-718, areutilized, the disclosure of which is incorporated herein by reference.

The disclosed system and method is in part a synthesis of recentadvances in understanding the physiology of aerosol generation by thehuman lung, specialized aerosol sampling and optical particle countingtechnology, combined with novel IqPCR technology capable of detection ofcytokines at attomolar levels. According to the disclosed methodologies,the IqPCR assays measure cytokines in the low femtogram, and morepreferably attogram, quantities per sample (1000 to 100 molecules persample). Exhaled droplet numbers and size distribution are counted withan optical particle counter designed for breath sampling, measuring lungvolumes, especially closing volume, and determining optimal methods forgenerating large numbers of exhaled droplets from the lower respiratorytract. Exhaled respiratory droplets may be collected with conventionalbreath condensate and aerosol filter methods (e.g., such as disclosed inMcKenzie J H et al. (2012) “Collection of Aerosolized Human CytokinesUsing Telfon® Filters,” PLoS ONE, Vol. 7, Issue 5, e35814), and thenassayed for cytokines in the samples using IqPCR. Cytokine measurementsare normalized with total droplet volume determined from opticalparticle count data. Exhaled breath measurements of respiratory fluidcytokine concentrations are then validated in three groups of subjects,including subjects with COPD, lung cancer, and normal or healthy lungs,by examining concordance with cytokine concentrations in bronchoalveolarlavage fluid (BAL) fluid determined by conventional methods.

The disclosed system and method revolutionizes the field of exhaledbiomarker measurement by substantially increasing the sensitivity ofbiomarker assay, while also increasing the release of biomarkers fromthe lung by obtaining samples while the subject is in a supine positionwhen performing breathing maneuvers. As a result, the efficiency of bothcollection methods and assay sensitivity is greatly improved.

Understandings about the physiology of aerosol droplet generation by thelung are utilized in order to increase the output of respiratory fluiddroplets. The fundamental features of “airway closure” were described byDollfuss and colleagues (Dolifuss R E et al. (1967) “RegionalVentilation of Lung Studied with Boluses of 133XENON,” RespirationPhysiology 2:234). The lung volume at which airway closure begins duringa progressive slow exhalation is termed the closing point (CP), and thevolume remaining to residual volume is termed the closing volume (CV).The terminal bronchioles are generally considered the site of airwayclosure. With airway closure, respiratory tract lining fluid (RTLF) inthe terminal bronchiole forms films that rupture when the airwayreopens, thereby causing particles to form.

Airway closure maneuvers that involve exhalations to low-lung volumes(near residual volume or RV) may be employed, such that a portion of theterminal bronchioles collapse, temporarily closing the airways. Theterminal bronchioles are then reopened upon the next inhalation (seeAlmstrand A et al. (2010) “Effect of airway opening on production ofexhaled particles,” J. Appl. Physiol. 108:584-8; Schwarz K et al. (2010)“Characterization of exhaled particles from the healthy human lung—asystematic analysis in relation to pulmonary function variables,” J.Aerosol Med. Pulm. Drug Deliv. 23:371-9; Holmgren H et al. (2010) “Sizedistribution of exhaled particles in the range from 0.01 to 2.0 mu m,”J. Aerosol Sci. 41:439-46). Studies have shown that deep exhalationresults in a four- to more than ten-fold increase in concentration, andrapid inhalation may produce a further two- to three-fold increase inconcentration (see Fabian P et al. (2011) “Origin of exhaled breathparticles from healthy and human rhinovirus-infected subjects,” J.Aerosol Med. Pulm. Drug Delivery, In Press; Johnson G R and Morawska L(2009), supra., J. Aerosol Med. Pulm. Drug Deliv.; Schwarz K et al.(2010), supra., J. Aerosol Med. Pulm. Drug Deliv. 23:371-9; Papineni R Sand Rosenthal F S (1997), supra., J. Aerosol Med. 10:105-16).

In contrast, rapid exhalation has little effect on the measuredconcentration. Droplet formation occurs in the lower airways as a resultof dynamic airway closure, which upon reopening destabilizes the mucouslining of the lower airways, thereby producing tiny droplets whichcontain submicron particles from the lower airways (Johnson G R andMorawska L (2009), supra., J. Aerosol Med. Pulm. Drug Deliv.; AlmstrandA et al. (2010), supra., J. Appl. Physiol. 108:584-8; Schwarz K et al.(2010), supra., J. Aerosol Med. Pulm. Drug Deliv. 23:371-9; Edwards D Aet al. (2004), supra., Proc. Natl. Acad. Sci. USA 101:17383-8).

In the supine position, because of abdominal contents pushing on thediaphragm, the closing volume is closer to and may exceed the functionalresidual capacity (FRC). Thus, breathing while in a supine position isbelieved to increase the output of exhaled droplets of respiratorylining fluid. Thus, in light of this phenomenon, production of RTLFdroplets may be further enhanced by combining supine posture withexhalation to RV. Such a breathing maneuver and position of the subjectis utilized to increased airway closure and generation of nanodropletsfrom respiratory fluid, and thus exhaled nanodroplets from the deeplung.

In addition, rather than collect droplets by inefficient, conventionalcondensation methods, methods adapted from air pollution monitoring areutilized to collect all expired droplets with high efficiency, such asTEFLON® filters (see McKenzie J H et al. (2012), supra., PLoS ONE, Vol.7, Issue 5, e35814, the disclosure of which is incorporate herein byreference).

In one embodiment, a human exhaled nano-droplet collector such asdisclosed in U.S. Pat. No. 8,250,903 is utilized in combination with themethodologies of the present invention. In particular, samplescollecting using such collector device are then assayed using IqPCR, asdiscussed in further detail below.

In other embodiments, a human exhaled nano-droplet collector is utilizedwith enhanced impaction of collected particles into a liquid medium.Such collection method exhibits >85% collection efficiency at 50 nm intoliquid medium. A liquid-impinger collection device is provided, whichcollects aerosolized particles into a liquid medium through inertialimpaction. The use of liquid collection medium prevents desiccation andpossible degradation of collected particles. Conventional liquid impingedevices suffer from various disadvantages, such as having a relativelylow sample flow rate and high sample collection volume, resulting inconsiderable dilution of collected particles. The collection device ofthe present invention, used in combination with IqPCR, substantiallyreduces sample collection volume required.

An exemplary collection system according to an embodiment of the presentinvention is illustrated in FIG. 1. The system includes a cone-shapedinlet for collection of exhaled breath from a subject. The shape andsize of the inlet permits the subject to wear a mask or respiratorduring testing. A seating area is provided for the subject, which may betented or enclosed. A humidifier is provided within the tented area inorder to increase moisture content therein. A 5.0 μm slit inertialimpactor collects particles greater than 5 μm. A steam generator, andassociated pump and water reservoir, is provided. The steam generatorinjects a turbulent flow of steam into the flow path in order toincrease the relative humidity to near saturation at a relatively warmtemperature. This increase in relative humidity makes it possible tosubsequently create supersaturation of water vapor by rapidly coolingthe high humidity fluid flow using a chiller. In the chiller, watervapor condenses on the small particles in the fluid flow, therebycausing them to grow into larger water droplets which can then becollected downstream with an inertial impactor. Thus, condensation fromthe steam generator grows remaining particles to a size large enough tobe collected by the downstream impactor (e.g., a 1.0 μm slit inertialimpactor). The particles are carried along via a regenerative air pump,and ultimately collected in a filter.

In order to substantially lower the minimum detectable and quantifiablecytokine concentration by several thousand orders of magnitude, ascompared to conventional methodologies, IqPCR is utilized. Human exhaledbreath contains attoliter to femtoliter volume droplets that containrare protein biomarkers. The total collection over a 20 min samplingperiod contains but a few picoliters of these droplets. As a result ofthe small sample volume and relatively low concentration of thepotential biomarkers, a subject sample may have at most thousands ofprotein biomarker molecules, and more typically tens to hundreds ofmolecules per sample. When diluted by a factor of 10⁶ into a typicalvolume for an assay (i.e. 50 μL), the resulting attomolar concentrationsof most biomarkers are not detectable using conventional immunoassaytechniques, such as ELISA or the Luminex assay (wherein typicaldetection limits range from nanomolar to femtomolar). The utilization ofIqPCR for the detection of biomarker proteins in exhaled breathleverages the well-established nucleic acid amplification technique ofPCR in order to provide amplification in an immunoassay (see Adler M etal. (2008) “Sensitivity by combination: immuno-PCR and relatedtechnologies,” Analyst 133:702-718; Niemeyer C M et al. (2005)“Immuno-PCR: high sensitivity detection of proteins by nucleic acidamplification,” Trends Biotechno. 23:208-216; Niemeyer C M et al. (2007)“Detecting antigens by quantitative immuno-PCR,” Nat. Protoc.2:1918-930). As noted above, IpPCR is substantially more sensitive thanconventional techniques, providing a 10- to 1000-fold increase insensitivity compared to techniques such as ELISA and mass spectrometryassays. For example, according to a study directed to an IqPCR methodfor detection of Staphylococcus aureus enterotoxin B (SEB), the IqPCRwas approximately 1000 times more sensitive (<10 pg ml⁻¹) when comparedto ELISA using the same couple of capture-detection antibodies (RajkovicA (2006) “Immunoquantitative real-time PCR for detection andquantification of Staphylococcus aureus enterotoxin B in foods,” Appl.Environ. Microbiol., 72(10):6593-6599). In that study, the assayconsisted of immunocapture of SEB and real-time PCR amplification of theDNA probe linked to the detection antibody (Id.).

The immunoassay portion of IqPCR utilized in the present inventionresembles in part conventional techniques. Referring to FIG. 2, thesample is applied to a substrate (e.g., microbeads), onto which isimmobilized an antibody directed toward the biomarker of interest. Afterwashing, a recognition antibody is applied to target any biomarkerproteins captured at the surface. However, whereas ELISA uses an enzymelabeled immunorecognition system, IqPCR uses a custom-synthesizedDNA-labeled antibody. As a result, a DNA template marks the capturedbiomarkers in the assay. The DNA is then amplified using qPCR, whichprovides several orders of magnitude gain, enabling the detection ofvery low numbers of biomarker antigens (see Barletta J M et al. (2005)“Detection of ultra-low levels of pathologic prion protein in scrapieinfected hamster brain homogenates using real-time immuno-PCR,” J.Virol. Methods 127:154-64; Barletta J et al. (2009) “Immunomagneticquantitative immuno-PCR for detection of less than one HIV-1 virion,” J.Virol. Methods 157:122-32; Burbulis I et al. (2005) “Using protein-DNAchimeras to detect and count small numbers of molecules,” Nat. Methods2:31-7; Burbulis I et al. (2007) “Quantifying small numbers ofantibodies with a ‘near-universal protein-DNA chimera,” Nat. Methods4:1011-3; Adler M et al. (2003) “A real-time immuno-PCR assay forroutine ultrasensitive quantification of proteins,” Biochem. Biophys.Res. Commun. 308:240-50).

In addition, the IqPCR technique is well suited for the disclosedmicrofluidic devices. Such devices may provide for microfluidic dropletImmuno-qPCR for multiplexed detection of trace biomarkers, and thestandardization by volume of exhaled droplets. The microfluidic dropletIqPCR of the present invention provides for multiplexed detection oftrace biomarkers and quantities of proteins (<1000 total proteins). Bycomparison, current methodologies (e.g., ELISA) require millions ofproteins for detection. As such, the systems and methods of the presentinvention utilizing IqPCR provide an exponential amplification within animmunoassay, and thus extreme detection sensitivity. Single proteindetection is therefore possible using the disclosed IqPCR methodologies.

Conventional IqPCR assays are typically done in plates, and thus requirea tremendous amount of tedious manual effort. According to aspects ofthe present invention, chemical immobilization of antibodies into amicrofluidic chip is provided (FIGS. 3 and 4). In particular, a novel,multiplexed IqPCR assay in a microfluidic chip is provided, whichsimultaneously measures multiple targets in each sample. A multiplexedimmunoassay is provided within a microfluidic channel of themicrofluidic chip. Magnetic microbeads are utilized, which may be heldin place during reagent loading and rinses. A relatively high surfacearea is provided for antibody-antigen reaction as compared to wellplates or microfluidic channel walls. Thus, there is no need toimmobilize and pattern antibodies within the chip. Conventionalmultiplex assay systems (e.g., Luminex system) are not compatible withmultiplexed IqPCR—PCR product from each bead type must be kept separate.

With reference to FIG. 4, a microfluidic droplet IqPCR assay in amicrofluidic chip is provided via an on-chip thermo cycler. The dropletIqPCR provides for the multiplexed detection of various tracebiomarkers. Droplet PCR enables isolation of each bead during PCRreaction. Fluorescent droplets after thermal cycling indicates PCRamplification of immuno-label, wherein the particular antigen detectedby the microbead is indicated as A1, A2 and A3, and the microbeadsimpacted in liquid medium are shown as A1′, A2′ and A3′.

Having generally described the invention, the same will be furtherunderstood through reference to the following additional examples, whichare provided by way of illustration and are not intended to be limitingof the present invention unless specified.

Cytokine Aerosol Sampling and Recovery from Filters

Various collection methods were tested using generated aerosols of amixture of six human cytokines including: IFN-gamma, IL-1 beta, IL-7,IL-8, IL-13, and TNF-alpha. (see McKenzie J H et al. (2009) “Use ofTeflon filters improves collection efficiency of aerosolized cytokines,”Am. J. Respir. Crit. Care Med. 179:A1365; McKenzie J H (2010) “Methodsfor environmental endotoxin assay and respiratory biomarkermeasurement,” Lowell, Mass.: University of Massachusetts Lowell, Schoolof Health & Environment). Cytokines in nebulizer solutions and aerosolsamples were assayed using analyte detection LINCOPLEX™ kits (Millipore,Billerica, Mass.) using a Luminex 200 IS system (Luminex Corp. Austin,Tex.). Based on measurements of nebulizer contents before and afterexperiments, all cytokine aerosols were in the rage of 20-40 pg/m³.

Collection efficiency of two air-to-liquid samplers, the SKC BIOSAMPLER®(SKC Inc, Eighty Four, Pa.) and the OMNI-3000™ wet wall cyclone (SceptorIndustries, Kansas City, Mo.), was compared with that of Teflo 2.0micron TEFLON® filters (Pall Corp., East Hills, N.Y.). Cytokines wererecovered from filters by extraction in 1 mL of 1×PBS, 1% BSA with 0.01%Tween-20. Filter extracts were concentrated before immunoassay usingAmicon Ultra-4 3000 MWCO (Millipore, Billerica, Mass.) centrifugalultra-filtration membranes.

Overall yield from filter collection, extraction, and centrifugal filterconcentration was, on average for six cytokines, 26%. TEFLON® filtersproduced significantly higher yields (p<0.05) than did either of the airto liquid samplers (SKC BIOSAMPLER® 4.8%; OMNI-3000™, 5.6%). It isbelieved that the higher yields from filter extracts reflect that theconventional air-to-liquid samplers did not efficiently collect thesmall droplets generated by the nebulizer.

Exhaled Breath Respiratory Droplet Sampling and Analysis

In a study of influenza patients, an Exhalair system (Pulmatrix Inc,Lexington, Mass.), which measured particles using an optical particlecounter and determined particle concentrations, was utilized to collectparticle data on and filter samples of exhaled breath. The vast majorityof exhaled particles were found to be in the submicron range, with largeinter-individual variation in production rates. Influenza virus RNA,collected on TEFLON® filters, was associated with fine particles (seeFabian P et al. (2008), supra., Plos One 3). Particle generation wassubsequently studied from both healthy subjects and subjects infectedwith human rhinovirus. It was found that respiratory droplets appear tobe formed during inhalation, that rate of inhalation and exhalation havelittle impact, and that production is consistent within a person overtime but highly variable between individuals. Therefore, controlling forexhaled particle volume is an important mechanism for standardizingcytokines measured in exhaled breath samples.

By increasing particle output with deep exhalation, combined with thecollection of exhaled droplets on TEFLON® filters, the reliabledetection of exhaled cytokines was displayed (McKenzie J H (2010),supra., Lowell, Mass.: University of Massachusetts Lowell, School ofHealth & Environment). Exhaled breath of ten healthy volunteers wascollected three times for 20 minutes each. Filters were extracted andassayed using a 12-plex LINCOPLEX™ kit (Millipore, Billerica, Mass.), asdescribed above for the experimental cytokine aerosols. Exhaled particlevolume was estimated from optical particle counts (CI-550, ClimetInstruments, Redlands, Calif.) made before and after the filtercollection while subjects used the same breathing pattern as during thefilter collection.

Total estimated particle volume of exhaled particles/20 min ranged from0.87 to 178 picoliters, with a median of 48 pL. Amounts of IFN-y, IL-1β,IL-7, and IL-8 were above the limit of detection in ≧50% of samples.Intra-class correlations (ICCs) for these four cytokines were 0.02(IFN-γ) to 0.34 (IL-1β), using femotogram (fg) of cytokine measured persample without standardization by respiratory fluid volume. Afternormalization for particle volume, the ICCs increased and ranged from0.42 (IL-8) to 0.92 (IL-7), indicating that adjustment for respiratoryfluid volume allowed more reliable discrimination between subjects. Theresults indicate that exhaled droplet volume be used to standardizeexhaled biomarker measurements.

Immuno-qPCR Method

IqPCR was implemented for biomarkers including IL-8. Exhaled breath iscollected, and IL-8 concentration in exhaled breath with that assayedvia IqPCR in the bronchoalveolar lavage fluid (BAL) is compared in 10subjects with cancer, 10 subjects with COPD, and 10 healthy controls.BAL fluid is thought to be representative of the respiratory tractlining fluid from terminal bronchioles. A concordance of cytokineconcentrations from exhaled breath particles and BAL is sought.Physiological maneuvers shown to enhance production of respiratorydroplets are utilized.

Filter samples acquired from the Exhalair are utilized. Samples areextraction in 1 mL of 1×PBS, 1% BSA with 0.01% Tween-20. Extracts andbreath condensates are concentrated to 100 μl using Amicon Ultra-4 3000MWCO. To conduct the immunoassay, immuno-detection kits withimmuno-magnetic beads are utilized (i.e., magnetic microbeads withantibodies immobilized to the surface). The sample is combined with theimmuno-labeled beads (e.g., per manufacturer recommendations). The useof magnetic beads enables simple rinses, which are conducted after eachstep.

After incubating the sample with the beads and rinsing, biotin-labeleddetection antibodies are added, followed by the addition ofstreptavidin, which binds strongly with all biotin-antibodiesimmobilized on the beads. Then, biotin-labeled DNA template is added.Because streptavidin has four biotin binding sites, the DNA template isalso captured at beads that have been marked by the immuno-label.

For the DNA template, a 104 base-pair sequence primer from the p29plasmid is utilized. (See Barletta J M et al. (2005), supra., J. Virol.Methods 127:154-64; Barletta J et al. (2009), supra, J. Virol. Methods157:122-32). Finally, qPCR master mix is added, and qPCR is then run onthe sample. This enables an approximate quantification of the number ofbiomarker molecules that were in the exhaled breath samples.

Study Population

Subjects undergoing bronchoscopy as a part of diagnostic workup fortheir condition are recruited, as well as normal/healthy controls andCOPD/Asthma volunteers. All subjects are age 18 or older. All subjectsrecruited were able to perform the exhaled breath collection maneuversatisfactorily. Initial studies of lung volumes and exhaled breathparticle number were conducted on a convenience sample of personsundergoing routine lung function testing.

Pulmonary Physiologic Measurements and Exhaled Breath Collection

Spirometric measurement of forced expiratory volume at 1 second interval(FEV₁), forced vital capacity (FVC), and plethysmographic measurementsof total lung capacity (TLC), FRC and RV are performed (University ofMaryland Hospital, pulmonary function laboratory). Closing volume ismeasured by standard inert gas bolus methods in both sitting and supinepositions.

In initial experiments, three replicate measurements of exhaled particlenumbers were made in the sitting and supine positions, alternatingrandomly between positions on 20 subjects. For each particle countmeasurement, the subject was provided a 3-minute exhaled breath sampleusing the Exhalair. Each subject was instructed to wear a nose clip toprevent nasal and environmental contamination of samples, and instructedto breathe into a mouthpiece according to a specific pattern.

After an initial washout period, software prompts displayed on Exhalairscreen instructed each subject to breathe a repeating pattern of tidalbreathing at normal lung volumes for 60 seconds followed by an airwayclosure maneuver requiring repeated exhalation to residual volume for 30seconds.

In the primary study, each participant provided a particle countmeasurement with the Exhalair before and after providing threeconsecutive 20-minute breath samples collected on filters. Three EBCsamples were collected using the standard protocol specified for theR-Tube. A nose clip was worn for all count, filter, and EBC collections.Filters and condensate were stored at −80° C. until assayed.

BAL Collection and Analysis

Patients undergoing bronchoscopy and BAL as a part of their diagnosticworkup were recruited. In these patients, a small part of the diagnosticSAL sample was obtained. In healthy volunteers and COPD volunteers, thevolunteers underwent fiber-optic bronchoscopy with sedation withmidazolam and fentanyl and topical anesthetization of the nasopharynxand oropharynx with 2% lidocaine nebulization and jelly. Thebronchoscopy and bronchoalveolar lavage was performed and the samplesgiven a code.

The samples were then immediately taken to the lab, where the BAL wasfirst poured through sterile gauze. The cells were then spun down bycentrifugation at 1000 g for 10 minutes. The sample was frozenimmediately for further analysis. The supernatant and lysed cell pelletswere analyzed for cytokine protein level by Luminex assay, and resultsstandardized to the total protein in the supernatant and cell lysate,respectively.

Statistical Data Analysis

Data consist of the following elements: (a) duplicate particle counts infour size ranges and total particle volume measured in sitting andsupine positions for each participant in the initial experiments and inone posture for the main experiment; (b) triplicate filter samplecytokine quantification (total mass by immunoassay and concentrationnormalized as mass divided by total particle volume); (c) triplicate EBCcytokine mass, cytokine concentration in ESC fluid, and estimatednormalized by respiratory droplet volume; (d) a single quantification ofSAL cytokine concentration; and (e) a measurement of lung volumes.

ANOVA is utilized to analyze the relationship between particle counts inthe four size ranges and posture, controlling for lung volumes and age.The relationship of particle volume output to posture, age, and lungvolumes is analyzed using linear models, supplemented by generalizedadditive models as needed to account for non-linear relationships.Intra-class correlation coefficients are computed for both totalcytokine mass collected and cytokine concentration adjusted for volumeof expired droplets. Correlations between cytokine measurements from infilter samples, EBC samples and BAL samples are examined using bothtotal cytokine mass detected and cytokine concentration in EBC and inrespiratory droplets. All statistical computations are performed usingSAS (Carey, N.C.) and R (Vienna, Austria).

Discussion

The utilization of IqPCR with immunomagnetic beads may be utilized forthe detection of rare biomarker proteins in exhaled breath samples. Themeasurements have external validity by comparison with BAL.

Further, a highly innovative system for the multiplexed detection ofbiomarkers in exhaled breath is provided by utilizing a microdropletqPCR in a microfluidic chip. For the immunoassay, Luminex multi-analytebead kits are suitable. In these kits, magnetic beads are doped with twodifferent fluorophores, and the relative concentration of eachfluorophore is uniquely assigned to the antibody attached to that bead.After performing the DNA-labeled immunoassay, the sample is loaded ontoa microfluidic chip that performs microdroplet qPCR. In this technique,the microfluidic chip forms picoliter droplet emulsions from the sample(following the addition of master mix). qPCR is then performed while thedroplets are on the chip (see Markey A L et al. (2006) “High-throughputdroplet PCR,” Methods 50:277-81; Ottesen E A et al. (2006) “Microfluidicdigital PCR enables multigene analysis of individual environmentalbacteria,” Science 314:1464-7; Schaerli Y et al. (2009) “Continuous-flowpolymerase chain reaction of single-copy DNA in microfluidicmicrodroplets,” Anal. Chem. 81:302-6; Srisa-Art M et al. (2009)“High-throughput confinement and detection of single DNA molecules inaqueous microdroplets,” Chem. Commun (Cam b) 6548-50; Wang F and Burns MA (2009) “Performance of nanoliter-sized droplet-based microfluidicPCR,” Biomed. Microdevices).

SYSR-green fluorescent images of the droplet indicate whether qPCRoccurred within each droplet (a SYBR-green droplet indicates PCRamplification, and thus the presence of the biomarker). Because of thesmall volume of each droplet, statistically there will be fewer than onebead per droplet, and thus qPCR is performed on individual beads.Therefore, after SYBR-green fluorescent droplets are identified, thebead fluorescence within these drops may be determined. Because of thedesign of the Luminex beads, the analyte that was detected by IqPCRwithin that droplet can be identified.

The disclosed methodology may be utilized in the identification of otherbiomarkers for diagnosis and management of COPD, asthma, lung cancer,tuberculosis (including multi-drug-resistance tuberculosis), and otherdiseases and disorders.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A system for detecting biomarkers in exhaledbreath nanodroplets, comprising: a collection device configured tononinvasively collect exhaled breath condensate nanodroplets from asubject by impacting said nanodroplets into a liquid medium, saidcollection device comprising an optical particle counter configured todetermine number and size distribution of said nanodroplets; andimmuno-quantitative polymerase chain reaction (IqPCR) assay fordetecting one or more target biomarkers in said nanodroplets.
 2. Thesystem of claim 1, wherein said IqPCR assay comprises a microbead havingan antibody immobilized thereon, wherein said antibody binds to aparticular one of said target biomarkers.
 3. The system of claim 1,wherein said IqPCR exhibits at least a 10-fold increase in sensitivityin detection of said target biomarkers as compared to enzyme-linkedimmuno-sorbent assay (ELISA).
 4. The system of claim 3, wherein saidIqPCR exhibits at least a 1000-fold increase in sensitivity in detectionof said target biomarkers as compared to enzyme-linked immuno-sorbentassay (ELISA).
 5. The system of claim 1, wherein said biomarkers areassociated with a respiratory disease, disorder or infection.
 6. Thesystem of claim 1, wherein said biomarkers comprise cytokines.
 7. Thesystem of claim 6, wherein said biomarkers are selected from the groupconsisting of IFN-gamma, IL-1 beta, IL-7, IL-8, IL-13, and TNF-alpha. 8.The system of claim 1, wherein said IqPCR assay comprises a plurality ofmicrobead-antibody complexes for simultaneous detection of multipletarget biomarkers.
 9. The system of claim 1, wherein at least one ofsaid target biomarkers is associated with a respiratory disease,disorder or infection.